The heterochronic pathway in Caenorhabditis elegans controls the timing of developmental events. One member of this regulatory pathway is lin-4. The product of lin-4 is a phylogenetically conserved founding member a large class of small noncoding RNAs known as microRNAs (miRNAs). The expression of lin-4 is activated at a specific time during the worm's post-embryonic development and acts by repressing the translation of target mRNAs by hybridizing to their 3' untranslated region. Although nematode miRNAs are some of the best-characterized miRNAs to date, the elements and factors that govern the specificity of their temporal expression are unknown. Preliminary data from the Ambros laboratory suggest that the expression of lin-4 may be transcriptionally regulated. In addition, there is evidence from the Ambros lab that at least two previously uncharacterized proteins may be involved in the regulation of lin-4 in the worm. The objectives of this proposal are to characterize the role of these candidate proteins in modulating the expression of lin-4, and to identify and characterize cis-acting and other trans-acting regulatory elements that participate in temporal expression of lin-4 in C. elegans. Understanding how the expression of lin-4 occurs in the nematode can provide valuable insights on how animal development, including that of mammals, is regulated.